EP4130286A1 - Verfahren zur deamidierung von proteinen - Google Patents

Verfahren zur deamidierung von proteinen Download PDF

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Publication number
EP4130286A1
EP4130286A1 EP21781755.0A EP21781755A EP4130286A1 EP 4130286 A1 EP4130286 A1 EP 4130286A1 EP 21781755 A EP21781755 A EP 21781755A EP 4130286 A1 EP4130286 A1 EP 4130286A1
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Prior art keywords
protein
deamidation
reaction
enzyme
salt
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English (en)
French (fr)
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EP4130286A4 (de
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Keita Okuda
Kiyota SAKAI
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Amano Enzyme Inc
Amano Enzyme USA Co Ltd
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Amano Enzyme Inc
Amano Enzyme USA Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
    • A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
    • A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/185—Vegetable proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/19—Dairy proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/50—Hydrolases (3) acting on carbon-nitrogen bonds, other than peptide bonds (3.5), e.g. asparaginase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
    • C12Y305/01044—Protein-glutamine glutaminase (3.5.1.44)

Definitions

  • the present invention relates to deamidation of a protein. Specifically, the present invention relates to a protein deamidation method and uses thereof, an agent for modifying protein used in a protein deamidation method, and the like.
  • Patent Document 1 Japanese Patent Laid-open Publication No. 2000-50887
  • an object of the present invention is to provide a method for promoting a deamidation reaction by a protein-deamidating enzyme.
  • a first aspect of the present invention relates to a method for deamidating a protein (protein deamidation method).
  • the protein deamidation reaction is promoted by treating a protein material with a protein-deamidating enzyme in the presence of a salt and/or a polysaccharide.
  • the protein (substrate protein) to be subjected to the enzyme treatment in the present invention is not particularly limited as long as it is subjected to the action of the enzyme, and there is no particular restriction on its origin, property, and the like.
  • the protein may be any of a vegetable protein, an animal protein, a protein derived from a fungus or a bacterium, a protein derived from algae, and the like.
  • Examples of the vegetable protein include proteins derived from beans such as soybeans, green peas, lentils, chickpeas, black beans, and fava beans; proteins derived from cereals such as wheat, barley, oat, rye, and rice; proteins derived from nuts such as almond and peanut; and proteins derived from seeds such as hemp seeds, chia seeds, quinoa, and amaranthus.
  • beans such as soybeans, green peas, lentils, chickpeas, black beans, and fava beans
  • proteins derived from cereals such as wheat, barley, oat, rye, and rice
  • proteins derived from nuts such as almond and peanut
  • proteins derived from seeds such as hemp seeds, chia seeds, quinoa, and amaranthus.
  • the animal protein may be derived from either a mammal or an insect.
  • the protein derived from a mammal include milk proteins such as casein and ⁇ -lactoglobulin; egg proteins such as ovalbumin; meat proteins such as myosin and actin; blood proteins such as serum albumin; and tendon proteins such as gelatin and collagen.
  • milk proteins such as casein and ⁇ -lactoglobulin
  • egg proteins such as ovalbumin
  • meat proteins such as myosin and actin
  • blood proteins such as serum albumin
  • tendon proteins such as gelatin and collagen.
  • the protein derived from an insect include proteins derived from crickets.
  • proteins derived from a fungus or a bacterium examples include proteins derived from yeasts, proteins derived from filamentous fungi, and proteins derived from fungi (mycoproteins derived from mushrooms, etc.).
  • vegetable proteins and animal proteins are preferable as an object of deamidation, and vegetable proteins are more preferable.
  • the substrate protein may be a protein chemically partially decomposed by an acid, an alkali, or the like, a protein enzymatically partially decomposed by a protease or the like, a chemically modified protein by various reagents, a synthetic peptide, or the like.
  • the substrate protein may be used singly, or two or more substrate proteins may be used in combination.
  • the substrate protein as described above is subjected to a reaction in the form of a solution, slurry, or paste.
  • concentration thereof is not particularly limited, and the concentration may be determined according to the desired property and state of the target deamidated protein.
  • the reaction is not limited to an aqueous solution, and a solution, a slurry, or a paste that is in an emulsion with oil or fat may be subjected to the reaction.
  • a salt, a saccharide, a protein, a flavor, a moisturizer, a colorant, and the like may be added to the solution, slurry, or paste of the substrate protein as necessary.
  • the salt used in the present invention is preferably a water-soluble salt.
  • the kind of the water-soluble salt is not particularly limited, and examples thereof include inorganic salts such as sulfate, hydrochloride, nitrate, dioxide, phosphate, hydrogen phosphate, polyphosphate, thiocyanate, thiosulfate, carbonate, and hydrogen carbonate and organic acid salts such as acetate, tartrate, citrate, and sorbate.
  • the kind of metal atoms constituting the water-soluble salt is not particularly limited, and examples thereof include alkali metals such as sodium and potassium; alkaline earth metals such as magnesium and calcium; and other metals such as manganese, copper, and zinc. Among them, an alkali metal is preferable.
  • polyphosphate may be linear, branched, or cyclic but is preferably cyclic.
  • the degree of polymerization of phosphoric acid in polyphosphate is not particularly limited and is, for example, 2 to 1,000, preferably 3 to 100, more preferably 4 to 10, still more preferably 5 to 7.
  • polyphosphates hexametaphosphate is preferable.
  • Preferable examples of the salt used in the present invention include phosphate, hydrogen phosphate, polyphosphate, citrate, sorbate, and carbonate, and more preferable examples include potassium phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, trisodium phosphate, sodium polyphosphate, trisodium citrate, and sodium carbonate.
  • carbonate when carbonate is used as the salt, the deamination of the substrate protein can be performed remarkably efficiently, and further, the thermal stability of the protein-deamidating enzyme can be improved.
  • salts may be used singly, or two or more salts may be used in combination.
  • One example of a combination of two salts is a combination of hydrogen phosphate and carbonate.
  • the ratio thereof is not particularly limited, but for example, the amount of carbonate is 4 to 46 parts by weight, preferably 15 to 43 parts by weight, more preferably 20 to 41 parts by weight, per 1 part by weight of hydrogen phosphate.
  • the polysaccharide used in the present invention is preferably a water-soluble polysaccharide.
  • the kind of the water-soluble polysaccharide is not particularly limited, and examples thereof include starch, dextrin, dextran, cellulose, xylan, tamarind seed gum, guar gum, tara gum, locust bean gum, gum arabic, carrageenan, gellan gum, xanthan gum, karaya gum, pectin, polygalacturonic acid, and alginic acid.
  • These polysaccharides may be in the form of salts. Examples of the salt of the polysaccharides include alkali metal salts such as a sodium salt and a potassium salt.
  • acidic polysaccharides polysaccharides having a carboxy group in at least one of constituent monosaccharides
  • carrageenan polysaccharides having a carboxy group in at least one of constituent monosaccharides
  • gellan gum is more preferable
  • gellan gum is still more preferable
  • the protein-deamidating enzyme referred to in the present invention is an enzyme that deamidates an amide group of a glutamine residue and an asparagine residue in a protein, and examples thereof include the followings.
  • the deamidated protein has amphiphilicity and becomes an ideal surfactant, and the emulsifying power, emulsion stability, foaming property, and foam stability of the protein are greatly improved.
  • deamidation of a protein leads to improvement of various functional properties of the protein, and the use of the protein is dramatically increased.
  • the hydrophobicity of the protein is increased to change the higher-order structure of the protein.
  • protein-deamidating enzyme examples include protein-deamidating enzymes derived from the genus Chryseobacterium, the genus Flavobacterium, the genus Empedobacter, the genus Sphingobacterium, the genus Aureobacterium, and the genus Myroides. These protein-deamidating enzymes are disclosed in Japanese Patent Laid-open Publication No. 2000-50887 , Japanese Patent Laid-open Publication No. 2001-218590 , WO 2006/075772 , and the like.
  • enzyme that deamidates the side chain of a glutamine residue in a protein include protein glutaminase derived from the genus Chryseobacterium.
  • enzymes that deamidates an asparagine residue in a protein include protein-deamidating enzymes derived from the genus Luteimicrobium, the genus Agromyces, the genus Microbacterium, and the genus Leifsonia. These protein-deamidating enzymes are disclosed, for example, in WO 2015/133590 .
  • arginine deiminase derived from the genus Fusarium. This protein-deamidating enzyme is disclosed, for example, in WO 2008/000714 .
  • protein-deamidating enzymes include preferably protein glutaminase, more preferably protein glutaminase derived from the genus Chryseobacterium, still more preferably protein glutaminase derived from Chryseobacterium proteolyticum.
  • Protein glutaminase derived from Chryseobacterium proteolyticum is commercially available, for example, as Protein Glutaminase "Amano" 500 (manufactured by Amano Enzyme Inc.), and this commercially available product can be used.
  • protein-deamidating enzyme may be an enzyme modified by a protein engineering technique.
  • the protein-deamidating enzyme one prepared from a culture solution of a microorganism that produces the protein-deamidating enzyme can be used.
  • the microorganism to be used for the preparation of the protein-deamidating enzyme is not particularly limited but is a microorganism that produces the enzyme, and for example, a microorganism belonging to the genus Chryseobacterium, Flavobacterium, Empedobacter, Sphingobacterium, Aureobacterium, or Myroides can be used.
  • a microorganism into which the gene encoding the protein-deamidating enzyme has been introduced by genetic engineering may be used.
  • Specific examples of the microorganism suitable for preparation of the protein-deamidating enzyme include Chryseobacterium sp. No. 9670 belonging to the genus Chryseobacterium.
  • the protein-deamidating enzyme can be obtained from the culture solution or bacterial cell of the microorganism. That is, a secretory protein can be recovered from the culture solution, and the other proteins can be recovered from the bacterial cells.
  • a known protein separation and purification method centrifugation, UF concentration, salting-out, various types of chromatography using an ion exchange resin, etc.
  • a target enzyme can be obtained by centrifuging the culture solution to remove bacterial cells and performing a combination of salting-out, chromatography, and the like.
  • the bacterial cells are crushed by pressurization treatment, ultrasonic treatment, or the like and then separated and purified in the same manner as described above, whereby the target enzyme can be obtained.
  • the above-mentioned series of steps may be carried out after the bacterial cells are previously recovered from the culture solution by filtration, centrifugation, or the like.
  • the enzyme may be powdered by a drying method such as freeze drying or reduced pressure drying, or an appropriate excipient or drying aid may be used at that time.
  • the protein-deamidating enzyme used in the present invention it is desirable to use a purified protein-deamidating enzyme having a high purity, but the protein-deamidating enzyme may have any purity as long as it catalyzes a desired reaction.
  • these enzyme preparations may be those to which various salt, saccharide, protein, lipid, and surfactant are added as enzyme stabilizer.
  • the enzyme activity of protein glutaminase which is one of the protein-deamidating enzymes, is measured by the method described below using Z-Gln-Gly (Benzyloxycarbonyl-L-glutaminylglycine) as a substrate unless otherwise specified.
  • Deamidation of a protein is performed by incubating a reaction solution containing the protein, a salt and/or a polysaccharide, and a protein-deamidating enzyme.
  • the conditions of the treatment with the protein-deamidating enzyme are not particularly limited, and the protein concentration, the reaction temperature, the reaction pH, the reaction time, the addition amount of the salt (in the case where a salt is added), the addition amount of the polysaccharide (in the case where a polysaccharide is added), the addition amount of the enzyme (enzyme concentration), and the like may be adjusted through preliminary experiments to set optimal reaction conditions according to the enzyme to be used.
  • the concentration of the protein falls within, but is not limited to, 0.1 to 30 wt%, preferably 0.5 to 20% wt%, more preferably 0.9 to 11 wt% in the reaction solution.
  • the amount of the deamidating enzyme is 0.01 to 1,000 U, preferably 0.1 to 100 U, more preferably 0.5 to 20 U, still more preferably 1 to 10 U, per 1 g of the protein.
  • the concentration of the salt is 0.01 to 20% (W/V%), preferably 0.05 to 15% (W/V%), more preferably 0.1 to 11% (W/V%), in the reaction solution.
  • the concentration of the polysaccharide is 0.001 to 10% (W/V%), preferably 0.005 to 5% (W/V%), more preferably 0.01 to 1% (W/V%), in the reaction solution.
  • the reaction temperature is 5 to 80°C, preferably 20 to 70°C, more preferably 30 to 60°C.
  • the pH of the reaction solution is 2 to 10, preferably 4 to 8.
  • the reaction time is 10 seconds to 48 hours, preferably 10 minutes to 24 hours, more preferably 30 minutes to 12 hours, still more preferably 30 minutes to 4 hours.
  • a deamidated protein can be obtained under the above reaction conditions.
  • the conditions of these reactions are appropriately selected according to the physical properties of the protein and the salt, the polysaccharide, and the protein-deamidating enzyme to be used. The optimum reaction conditions may be determined through a preliminary experiment.
  • the present invention also provides a method for producing a deamidated protein and a method for producing a food or a pharmaceutical product containing a deamidated protein.
  • a typical aspect of the method for producing a deamidated protein includes the following steps (1) and (2).
  • a deactivation step of the protein-deamidating enzyme may be added after the step (2). Specific conditions of the following steps (1) and (2) are as described in the section "1.1. Protein Deamidation Method" above.
  • a typical aspect of the method for producing a food or a medicine includes the following steps (1) and (2).
  • a deactivation step of the protein-deamidating enzyme may be added after the step (2).
  • Specific conditions of the following steps (1) and (2) are as described in the section "1.1. Protein Deamidation Method" above.
  • Examples of the solution prepared in the step (1) include a solution prepared by adding a salt and/or a polysaccharide to vegetable milk prepared using a vegetable protein.
  • the deamidated protein produced by the method of the present invention has a commercial value by itself. On the other hand, it is also useful as an ingredient or a material for various foods and beverages. Therefore, the present application also provides a food or beverage containing the deamidated protein obtained by the production method of the present invention.
  • the food and beverage here are not particularly limited.
  • Examples of the food and beverage include processed marine products (tube-shaped fish paste cake, fish paste cake, fish minced and steamed, shredded and dried squid, dried fish, salted fish guts, a fish sausage, foods boiled in soy sauce, canned food, etc.), processed meat products (ham, bacon, sausage, jerky, corned beef, restructured meat, etc.), processed vegetable products (canned or bottled vegetable, processed tomato, processed mushroom, pickled vegetables, dried vegetables, vegetable boiled down in soy sauce, etc.), noodles and breads (various noodles, bread, a sweet roll, etc.), processed grains (cereal, oatmeal, muesli, processed rice, wheat-gluten bread, barley tea, etc.), dairy products (milk, processed milk, milk beverage, concentrated milk, powdered milk, condensed milk, fermented milk, lactic acid bacteria beverage, yogurt, butter, cheese, ice cream, etc.), processed fruit products (canned or bottled fruits, jam, marmalade, dried fruits, etc.),
  • the present invention further provides an agent for modifying protein that can be used for deamidation of a protein.
  • the agent for modifying protein of the present invention is typically used in the protein deamidation method, the method for producing a deamidated protein, or the method for producing a food or a medicine of the present invention.
  • the agent for modifying protein of the present invention contains a salt and/or a polysaccharide as active ingredients in addition to the protein-deamidating enzyme. Since the details of the protein-deamidating enzyme, the salt, and the polysaccharide are as described above, the description thereof will be omitted.
  • the content of the protein-deamidating enzyme falls within, but is not limited to, 1 to 2,000 U, preferably 10 to 1,500 U, more preferably 50 to 1,000 U, per 1 g of the preparation.
  • the content of the salt is 0.1 to 10 g, preferably 0.2 to 9 g, more preferably 0.5 to 8 g, per 1 U of the protein-deamidating enzyme.
  • the content of the polysaccharide is 0.01 to 10 mg, preferably 0.05 to 5 mg, more preferably 0.1 to 1 mg, per 1 U of the protein-deamidating enzyme.
  • the final form of the agent for modifying protein may be a liquid form or a solid form (including a powder form).
  • the agent for modifying protein may contain an excipient, a buffer, a suspending agent, a stabilizer, a preservative, an antiseptic, a physiological saline, and the like in addition to the active ingredients.
  • pea protein product name: NOW (registered trademark), manufactured by Sports Organic Pea Protein
  • pea protein product name: NOW (registered trademark), manufactured by Sports Organic Pea Protein
  • 10 wt% (final concentration) of pea protein, protein glutaminase (product name: Protein Glutaminase "Amano" 500, manufactured by Amano Enzyme Inc.), and 0.5% (W/V%) (final concentration) of sodium citrate were mixed, the pH was adjusted to 7.5 using hydrochloric acid or sodium hydroxide, and then the mixture was incubated at 52.5°C for 1.5 hours to cause a reaction.
  • ammonia liberated by protein glutaminase was quantified as follows.
  • the reaction solution was treated at 85°C for 10 minutes to stop the reaction, 1 mL of the reaction solution was centrifuged (13,000 rpm, 10 min), the obtained supernatant was diluted 100 times with ultrapure water, and ammonia was quantified using Ammonia Assay Kit (Sigma-Aldrich).
  • the amount of the enzyme was set to 7.5 U of protein glutaminase (final concentration) per 1 g of the substrate protein.
  • the deamidation rate was calculated by dividing the amount of ammonia liberated by protein glutaminase per 1 g of pea protein by the amount of ammonia liberated by acidolysis (reaction in 6-N hydrochloric acid (final concentration) at 110°C for 24 hours) of 1 g of pea protein. For comparison, the deamidation rate was measured by performing an enzyme reaction under the same conditions as described above except that sodium citrate was not added.
  • the influence of the treatment pH and the inorganic salt concentration was examined.
  • the reaction and evaluation were carried out in the same manner as in Example 2 except that pea protein was changed (in the present example, product name: PURIS Pea Protein 870, manufactured by PURIS Foods was used) and that the treatment pH and the concentration of dipotassium hydrogen phosphate (DKP) were changed.
  • the pH was adjusted using hydrochloric acid or sodium hydroxide.
  • a reaction between Benzyloxycarbonyl-L-glutaminylglycine (Z-Gln-Gly) and protein glutaminase was carried out using a solvent prepared by supplementarily adding sodium dihydrogen phosphate or sodium carbonate to a solution containing disodium hydrogen phosphate, and the influence of the supplementation of sodium dihydrogen phosphate or sodium carbonate on the deamidation reaction efficiency was evaluated.
  • the deamidation reaction efficiency obtained by performing a reaction between various substrate proteins and protein glutaminase using a solvent obtained by supplementarily adding sodium carbonate to a solution containing disodium hydrogen phosphate was evaluated. Specifically, egg albumin, milk casein, chick bean protein, pea protein, soybean protein, or wheat gluten (final concentration: 1.0 W/V%) was added to McIlvaine buffer (pH 7.0, containing 16.5 mM of disodium hydrogen phosphate and 1.8 mM of citric acid), and sodium carbonate (final concentration: 0.5 M) was further supplementarily added thereto to prepare a solution having a pH adjusted to 7.0.
  • an enzyme liquid containing 0.13 U/ml of protein glutaminase (product name: Protein Glutaminase "Amano" 500, manufactured by Amano Enzyme Inc.) was added, and a reaction was caused at 37°C for 20 minutes. Thereafter, 1.0 ml of a 0.4-M trichloroacetic acid solution was added to stop the reaction. Next, the amount of ammonia released by protein glutaminase was measured using an ammonia measurement kit (product name: Ammonia Test Wako, manufactured by FUJIFILM Wako Pure Chemical Corporation).
  • Fig. 2 The results are shown in Fig. 2 .
  • the deamination of the substrate protein by protein glutaminase is promoted when 0.5 M of sodium carbonate is supplementarily added to the solution containing disodium hydrogen phosphate.
  • the deamination reaction by protein glutaminase is promoted for both of the vegetable protein and the animal protein, and particularly, it has been revealed that the effect of promoting the deamination reaction is remarkably high for the vegetable protein.
  • the thermal stability of protein glutaminase was evaluated using a solvent obtained by supplementarily adding sodium carbonate to a solution containing disodium hydrogen phosphate.
  • protein glutaminase product name: Protein Glutaminase "Amano" 500, manufactured by Amano Enzyme Inc.
  • McIlvaine buffer pH 7.0, containing 41.2 mM of disodium hydrogen phosphate and 4.4 mM of citric acid
  • sodium carbonate final concentration: 1.0 M
  • the activity of protein glutaminase was performed by the method described above.
  • the residual activity value of protein glutaminase was determined with the activity value of protein glutaminase before incubation being 100%.
  • the test was performed under the same conditions as described above except that 1.0 M of sodium carbonate was not added, and the activity of protein glutaminase was measured over time.
  • the deamidation reaction is promoted, and the reaction efficiency can be expected to be improved.
  • the deamidated protein obtained by the present invention has a commercial value by itself and can also be used as an ingredient or material of various foods, beverages, or medicines. That is, the present invention is expected to be used and applied particularly in the food and beverage field and the pharmaceutical field.

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  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Coloring Foods And Improving Nutritive Qualities (AREA)
EP21781755.0A 2020-04-03 2021-04-02 Verfahren zur deamidierung von proteinen Pending EP4130286A4 (de)

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US11685914B2 (en) * 2017-09-07 2023-06-27 Amano Enzyme Inc. Stabilised dry protein deamidase composition
WO2023085316A1 (ja) * 2021-11-09 2023-05-19 天野エンザイム株式会社 組織化植物性タンパク質含有食品の製造方法
WO2023085315A1 (ja) * 2021-11-09 2023-05-19 天野エンザイム株式会社 植物性タンパク質含有組成物の消化性向上剤
CN118234866A (zh) * 2021-11-22 2024-06-21 天野酶制品株式会社 蛋白质脱酰胺酶
WO2023150033A1 (en) * 2022-02-02 2023-08-10 Corn Products Development, Inc. Edible compositions comprising deamidated legume protein isolates
WO2023149512A1 (ja) * 2022-02-03 2023-08-10 天野エンザイム株式会社 酵素製剤
JPWO2023190734A1 (de) * 2022-03-31 2023-10-05
CN114836403B (zh) * 2022-04-28 2023-08-25 江南大学 一种利用蛋白质谷氨酰胺酶改善糖酶催化性能的方法
WO2023219172A1 (ja) * 2022-05-12 2023-11-16 天野エンザイム株式会社 加工植物性タンパク質含有組成物の製造方法

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DE60021160T2 (de) 1999-12-03 2006-05-11 Amano Enzyme Inc., Nagoya Protein-deamiderendes Enzym, dafür kodierendes Gen, enzym-produzierendes Mikroorganismus, Verfahren zur Herstellung und Verwendung davon
WO2006075772A1 (ja) 2005-01-13 2006-07-20 Ajinomoto Co., Inc. 乳製品及びその製造方法
US20090297689A1 (en) 2006-06-26 2009-12-03 Luppo Edens Peptidylarginine deiminase and uses thereof in the production of citrullinated proteins and peptides
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JP6569662B2 (ja) 2014-03-07 2019-09-04 味の素株式会社 新規タンパク質脱アミド酵素
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CN115380116A (zh) 2022-11-22

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